Histidine-tag-directed chromophores for tracer analyses in the analytical ultracentrifuge

Lance M. Hellman, Chunxia Zhao, Manana Melikishvili, Xiaorong Tao, James E. Hopper, Sidney W. Whiteheart, Michael G. Fried

Research output: Contribution to journalReview articlepeer-review

10 Scopus citations

Abstract

Many recombinant proteins carry an oligohistidine (His. X)-tag that allows their purification by immobilized metal affinity chromatography (IMAC). This tag can be exploited for the site-specific attachment of chromophores and fluorophores, using the same metal ion-nitrilotriacetic acid (NTA) coordination chemistry that forms the basis of popular versions of IMAC. Labeling proteins in this way can allow their detection at wavelengths outside of the absorption envelopes of un-modified proteins and nucleic acids. Here we describe use of this technology in tracer sedimentation experiments that can be performed in a standard analytical ultracentrifuge equipped with absorbance or fluorescence optics. Examples include sedimentation velocity in the presence of low molecular weight chromophoric solutes, sedimentation equilibrium in the presence of high concentrations of background protein and selective labeling to simplify the assignment of species in a complex interacting mixture.

Original languageEnglish
Pages (from-to)31-38
Number of pages8
JournalMethods
Volume54
Issue number1
DOIs
StatePublished - May 2011

Bibliographical note

Funding Information:
This work was supported by NIH Grants GM27925 to J.E.H., NS-046242 and HL-566252 to S.W.W. and GM-070662 to M.G.F.

Keywords

  • Analytical ultracentrifugation
  • Fluorescence
  • Histidine tag
  • Nitrilotriacetic acid
  • Tracer sedimentation

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology (all)

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